Cysteine protease inhibitors for use in treatment of IGE mediated allergic diseases

ABSTRACT

The invention provides compounds for use in the treatment of allergic diseases including juvenile asthma and eczema. The compounds can inhibit IgE mediated reaction to major environmental and occupational allergens and can also have a prophylactic effect against allergic disease by preventing allergic sensitization to environmental and occupational allergens when administered to at-risk individuals (e.g., those at genetic risk of asthma and those exposed to occupational allergens in the workplace). The compounds are also useful for inactivation or attenuation of the allergenicity of allergens in situ. The invention provides compounds and ligands per se, pharmaceutical compositions containing the compounds, processes for producing the compounds and pharmaceutical compositions, and methods for using the compounds and compositions in treatment or prophylaxis of IgE mediated allergic diseases and in inactivation or attenuation of allergens in situ. The invention also enables the reduction or destruction of the viability of allergy-causing organisms.

FIELD OF THE INVENTION

The invention relates to compounds for use in the treatment of allergicdiseases including juvenile asthma and eczema.

Compounds of the invention can inhibit IgE mediated reaction to majorenvironmental and occupational allergens. They can also have aprophylactic effect against allergic disease by preventing allergicsensitisation to environmental and occupational antigens whenadministered to at-risk individuals (e.g. those at genetic risk ofasthma, and those exposed to occupational allergens in the workplace).The compounds of the invention can also be useful for inactivation orattenuation of the allergenicity of allergens in situ. The inventionprovides novel compounds and ligands per se, pharmaceutical compositionscontaining the compounds, processes for producing the compounds andpharmaceutical compositions, and methods for using the compounds andcompositions in treatment or prophylaxis of IgE mediated allergicdiseases and in inactivation or attenuation of allergens in situ. Theinvention also provides means for the reducing or destroying theviability of allergy-causing organisms.

The invention is made possible by our new understanding of the role ofthe low-affinity receptor for IgE (FceRII), also known as CD23, in IgEmediated allergic diseases.

DESCRIPTION OF THE RELATED ART

The Multiple Roles of CD23 CD23 plays an important role in theregulation of immune responses--particularly the regulation of IgEresponses. CD23 is a cell surface protein which extends from the plasmamembrane via a stalk which is cleaved proteolytically during immuneresponses. We have demonstrated that CD 23 is cleaved by Der p I; aprotease which is the major allergen of the house dust mite, althoughthe endogenous proteases responsible or cleaving CD 23 have not so farbeen identified. In its membrane bound form, CD23 acts as a cellularreceptor for IgE and is found on various cell types including B cells, Tcells, platelets, eosinophils, keratinocytes and also on antigenpresenting cells (including follicular dendritic cells) which presentantigens to T and B lymphocytes. The level of expression of CD23 at thecell surface determines its functionality and is regulated by cytokines,notably IL4.

In its membrane bound form, CD23 allows eosinophils to attach toparasites via antigen-specific IgE. It also plays an importantregulatory role on B lymphocytes (which produce antibodies). In thepresence of soluble IgE, probably in the form so immune complexes withthe allergen, cell surface CD23 becomes occupied by IgE, conveying aninhibitory signal to the B-lymphocyte. This is believed to be animportant negative feedback loop in the regulation of IgE synthesis.Occupancy of membrane bound CD23 by IgE protects CD23 from proteolyticcleavage, preventing the release of "cytokine active" forms of solubleCD23 (see below) which favour the production of IgE as opposed to otherclasses of immunoglobulin. CD23 also interacts with ligands (or"counterstructures") other than IgE. By association of CD23 on anactivated B cell with CD21 (the type two complement receptor "CR2") on afollicular dendritic cell of the lymph node, cell-surface CD23 functionsas an intercellular adhesion molecule. This function of CD23 is believedto be important in the rescue of germinal centre B lymphocytes from"apoptosis" (i.e. programmed cell-death), allowing the survival ofantibody producing clones which would otherwise have been destined todie. There is also evidence that CD23 associates with the cell-surfacemolecules responsible for presenting antigenic peptides to T lymphocytes(i.e. the HLA class-II molecules) and may thereby influence antigenpresentation to T lymphocytes. Moreover, the presence and degree ofexpression of CD23 on Langerhans cells (a type of antigen presentingcell), and its affinity for immune complexes comprised of allergen andIgE, will also determine to what extent such complexes are processed andpresented to T lymphocytes. CD23 may therefore influence antigenpresentation to both B and T lymphocytes, processes which determine thedegree and nature of immune responsiveness to foreign antigens.

Proteolytic cleavage of CD23 Native CD23 (45 ka) can be cleaved from thecell surface by proteolytic digestion at several sites within the stalkregion to generate soluble CD23 (sCD23). The largest soluble fragment isof 37 kDa. Cleavage nearer the membrane-distal lectin domain givessoluble fragments of 33, 29 and 25 kDa containing the lectin domain anda C-terminal tail. Some forms of sCD23 (notably the 37 kDa form) areactive upon ocher cells. Thus, Ghadieri et al and Bonnefoy et al havedemonstrated that sCD23 (37 kDa) is a potent stimulator of mastcells--eliciting degranulation at nanograms per ml concentrations.Moreover, the larger forms of sCD23 also have cytokine activities whichfavour the production of IgE and IgG4 subclass antibodies associatedwith allergic, anti-parasitic and chronic immune responses. Indeed invitro experiments have shown that in the presence of IL4 sCD23 inducesIgE producing B cells to differentiate into plasma cells (Liu; Gordon).The regulatory role of CD23 upon IgE synthesis has also been confirmedin vivo using antibodies to CD23 (which inhibit antigen-specific IgEresponses), and using CD23 gene-knockout mice, in which antigen-specificIgE responses are exaggerated.

In addition to these data from experiments in animals elevated levels ofsCD23 and of CD23 positive peripheral blood lymphocytes have beenreported in atopic individuals (Gordon et al) (Ghadieri et al)implicating CD23 as an important regulatory factor in IgE immuneresponses.

From these considerations it its evident that CD23 has importantregulatory functions determining the quality and quantity of an immuneresponse, particularly affecting humoral immunity (i.e. the productionof specific antibodies). Moreover, the physical form of CD23 (i.e.cellular versus soluble) has a major influence on its regulatoryfunction, particularly in the case of the IgE responses of Blymphocytes. Thus, CD23 in its cellular form participates in thenegative feedback inhibition of IgE synthesis. By contrast, in itssoluble forms CD23 stimulates the production of IgE via its cytokineactivities. The balance between cellular and soluble forms of CD23 istherefore seen to have a pivotal role in determining the character of animmune response, in particular whether IgE is produced against aparticular antigen, and also how much IgE is produced. However, thenature of proteases which bring about the cleavage of CD23 and whichdetermine the balance between membrane bound and soluble forms has notso-far been established, although current theory, supported only bycircumstantial evidence, has it that CD23 is autocatalytic, and bringsabout its own cleavage from the plasma membrane.

Proteolytic activity of certain environmental and occupationalallergens. Studies in mice and in man suggest that allergic sensitisingpotency of environmental allergens is, in some cases, related to theirproteolytic activity. Thus, papain (a cysteinyl protease of papaya) is apotent allergen in man. Also, inhaled bromelain (a cysteinyl protease ofpineapple) causes occupational allergies and asthma (Gailhoffer 1988).Also, the major allergen of house dust mite (Der p I), to which manyasthmatic individuals are sensitive, has proteolytic activity.Proteolytic enzymes of environmental antigens and parasites mayinfluence the quality of T lymphocyte responses to favour IgE production(reviewed by Finkelmann 1992) although how they do so has not beenestablished. Thus, subcutaneous daily injections of certain strains ofmice with papain result in a dramatic increase in non-specific IgE whichis markedly attenuated by prior inactivation of the catalytic activityof the enzyme. The elevation of total IgE by active papain is associatedwith the production of cytokines characteristic of the T_(k) 2 subset ofT-helper lymphocytes which are involved in allergic and anti-parasiticresponses. However, the substrate of this proteolytic mechanism wherebypapain elevates IgE production has not been identified.

SUMMARY OF THE INVENTION

Current teaching has it that CD23 is cleaved from the plasma membrane bya putative autoproteolytic activity (i.e. a proteolytic activity of CD23itself upon CD23 as a substrate). No candidate protease (eitherendogenous or exogenous) other than CD23 has been proposed. Moreover,the putative "autoproteolytic" activity of CD23 has never beendemonstrated. Surprisingly therefore, it has now been found that theexogenous protease and allergen Der p I, in highly purified form, isvery effective and specific at cleaving CD23 from the plasma membrane ofcultured B lymphocytes. Since the cleavage of CD23 is an importantregulatory step governing IgE synthesis, it follows that the potentallergic sensitising activity of Der p I (i.e. its allergenicity)resides, in part, in its ability to cleave CD23 from the cell surface.Although it had been speculated previously that the proteolytic activityof Der p I might be related to its allergenicity, no explanation hadbeen offered for the mechanism of the putative proteolytic event, norhas any candidate previously been proposed as a substrate of thisproteolytic activity.

In a first aspect of the invention, we have demonstrated that thecleavage of CD23 by (Gordon et al) is: i) stimulated by cysteine; ii)inhibited by the specific cysteinyl protease inhibitor E64; and iii) notinhibited by the trypsin protease inhibitor alpha-1-antitrypsin (whichinhibits various trypsin-like proteases as well as trypsin). Thecompound E64 is L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane(Sigma, Poole, UK).

These findings demonstrate that Der p I is indeed a cysteinyl proteaseas suggested tentitively by earlier studies, and moreover that it is thecysteinyl protease activity of Der p I which is responsible for CD23cleavage.

From these considerations, it follows that compounds of this inventionother than E64, yet capable (like E64) of inhibiting cysteinylproteases, would also prevent the cleavage of CD23 by Der p I. Thiswould include the peptide sequence comprising the cleavage sites of CD23which are cleaved by Der p I, and analogues thereof. The latter wouldinclude D-amino acid analogues, including "reverse-D" peptides madeexclusively of D-amino acids but of the reverse sequence of the naturalcleavage site--as described recently by Van Regenmortal et al forbiologically active analogues of CD4.

In a second aspect of the invention, we have now also demonstrated thatthe protease inhibitor human alpha-1-antitrypsin, rather than inhibitingDer p I, is a substrate for Der p I becoming cleaved at a specific site.Since Jul. 17, 1995, this site has been identified as "QVS/SGF"(Kalsheker N. et al 1996). It follows that peptide analogues (asdescribed above for CD23 cleavage sites) and non-peptide mimetics ofthis site may also be specific inhibitors of Der p I. Such compounds ofthis invention may therefore have uses as described above for inhibitorsof Der p I (i.e. prevention of in vivo cleavage of CD23 by Der p I).Moreover, since Der p I is an extracorporeal digestive enzyme of thehouse dust mite, it follows that inhibitors of Der p I may cause thedust mite to have "indigestion" (i.e. nutritional deprivation) due tothe failure of this enzyme. Indeed, since the food of the house dustmites is comprised mainly of human skin flakes (which containalpha-1-antitrypsin) it may be necessary to Der p I to destroy orinactivate alpha-1-antitrypsin in order to digest the skin flakes. Thus,in a third aspect of the invention inhibitors of Der p I are predictedto have a "toxic" effect (via nutritional deprivation) on house dustmites.

Inhibitors of Der p I may be useful for killing house dust mites insitu, in addition to attenuating the allergenicity (i.e. sensitisingactivity) of Der p I. We believe that these effects would synergiseresulting in a highly effective anti-asthma agent for application tofurnishings (beds, carpets etc.) which are the natural habitat for housedust mites.

We have demonstrated that the principal cleavage fragment of the native45 kDa form of CD23 released by Der p I is indistinguishable (by SDSelectrophoresis) from the major naturally occurring cleavage fragment ofCD23: i.e. the 25 kDa fragment. However, sequence analysis of theN-terminal of the fragment liberated by Der p I demonstrates that thecleavage site "QVS/SGF" recognised by Der p I is distinct from thenatural cleavage site that generates the 25 kda fragment. Smalleramounts of larger (presumably "cytokine active") forms of CD23 were alsogenerated by Der p I, indicating the existence of additional, moremembrane proximal cleavage sites in the stalk region. A further cleavagesite has also been identified by us in the C terminal tail region asSAE/SMG.

Since inhibitors of the CD23 cleavage activity of Der p I (such as E64and analogues) may also inhibit the endogenous protease(s) that cleaveCD23, whether or not these proteases are identical in specificity to Derp I the invention therefore includes inhibitors of endogenous proteasesthat cleave CD23 in addition to exogenous proteases such as Der p I andbromelain and certain other environmental allergens with proteolyticactivity. Where legally permissible, the invention includes the use ofinhibitors of the enzymatic cleavage of CD23 (whether by endogenous orexogenous proteases) for the treatment of allergic diseases such asjuvenile asthma and eczema, and the use of such inhibitors to inactivatethe proteolytic activities of environmental sensitising agents orallergens such as Der p I and bromelain.

In a third aspect the invention provides novel compounds which havecysteinyl protease inhibitor activity and are capable of inhibitingproteolytic cleavage of membrane bound CD23 in vivo excludingL-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E64).

In a forth aspect the invention provides cysteinyl protease inhibitorcompounds which include a chemical composition capable of adopting astructure essentially equivalent to an inhibitor of the enzyme Der p I,excluding E64, optionally together with a pharmaceutically acceptablecarrier or excipient for use in the treatment of allergic diseases.

In a fifth aspect the invention provides cysteinyl protease inhibitorcompounds capable of adopting a structure having a pharmacophoricpattern essentially equivalent to the pharmacophoric pattern of asection of an inhibitor of Der p I, excluding E64.

In a sixth aspect the invention provides a ligand which cross reactswith a cysteinyl protease inhibitor compound which inhibits the enzymeDer p I, excluding E64, which compound includes 1 or more copies of amotif which comprises:

i) a hydrogen bond donor;

ii) three hydrophobes; and

iii) a hydrogen bond acceptor.

In a seventh aspect the invention provides compounds or ligands of thegeneral formula (I): ##STR1## wherein X, Y and Z are N or CH;

R₁ is a blocking group for the N-terminal nitrogen;

R₂, R₃, and R₄ are side-chains on X, Y, and Z; and

W is a group that reacts irreversibly with an active cysteine thiol ofDer p I.

In a eighth aspect the invention provides an agent for treatment of IgEmediated allergic disease which includes as active ingredient aneffective amount of a compound selected from the group consisting of: acysteinyl protease inhibitor; a substrate for Der p I which reacts withDer p I at a specific site; and a Der p I inhibitor capable ofinhibiting the proteolytic enzyme activity of Der D I, the agentoptionally including one or more of a pharmaceutically acceptablecarrier, adjuvant or excipient.

In a ninth aspect the invention provides an agent for attenuating orinactivating the allergenicity of Der p I which includes as activeingredient an effective amount of a compound having Der p I inhibitoractivity, the agent optionally including one or more of a carrier,adjuvant, excipient.

In a tenth aspect the invention provides an agent for reducing ordestroying the viability of house dust mites which includes as activeingredient an effective amount of a compound having Der p I inhibitoractivity, the agent optionally including one or more of apharmaceutically acceptable carrier, adjuvant, excipient.

In an eleventh aspect the invention provides a process for producing acompound or ligand of the invention which comprises synthesising acysteinyl protease inhibitor compound or ligand and optionallyconjugating said compound or ligand to a carrier

Therefore, in summary, the present invention is based upon ourappreciation that the major allergen of house dust mite faeces (Der pI), is capable of cleaving CD23 (the low affinity receptor for IgE) fromthe cell-surface of B-lymphocytes and presumably from other cell types.We demonstrate that this activity is stimulated by cysteine and can beabolished by the well-known cysteinyl protease inhibitor E64.

The invention relates particularly to compounds capable of inhibitingthe proteolytic cleavage of CD23 from the plasma membrane of cells byexogenous proteases (such as Der p I) bromelain and certain proteasesand parasites) and to compounds capable of inhibiting endogenousproteases which cleave CD23 from the cell.

The compounds may also have a prophylactic effect against allergicdisease--by preventing allergic sensitisation to environmental andoccupational antigens when administered to at-risk individuals (e.g.those at genetic risk of asthma, and those exposed to occupationalallergens).

The compounds may also be used for the inactivation of the proteolyticactivity of environmental allergens in situ (e.g. house dust mite faecalallergen Der p I in beds, carpets and vacuum cleaners). Inactivation ofthe proteolytic activity of these allergens may attenuate theirallergenicity (i.e. their capability to provoke allergies and asthma)which is due to their capability to cleave CD23 from the cell-surface.

The compounds may also kill house dust mites by nutritional deprivation.

The present invention will now be described by way of non-limitingexamples only, with reference to FIG. 1 to 18 in which:

FIG. 1 shows CD23 expression by RPMI 8866 human 3 cells using FITClabelled mouse monoclonal anti-CD23 antibody;

FIG. 2 shows that the proteolytic effect Der p I is specific for CD23;

FIG. 3 shows that Der p I preferentially cleaves CD23 close to thelectin domain.

FIG. 4 shows the pharmacophore of the cysteinyl protease Der p I;

FIG. 5 shows the distance constraints of the pharmacophore of FIG. 4;and

FIG. 6 shows the angle constraints of the pharmacophore of FIG. 4.

FIG. 7 shows the pharmacophore and illustrates how compound 8 fits thepharmacophore.

FIG. 8 shows the pharmacophore and illustrates how compound 40 fits thepharmacophore.

FIG. 9 shows the pharmacophore and illustrates how compound 25 fits thepharmacophore.

FIG. 10 shows the distance and angle constraints between points 1-4-5 ofthe pharmacophore of FIG. 4.

FIG. 11 shows the distance and angle constraints between points 1-2-4 ofthe pharmacophore of FIG. 4

FIG. 12 shows the distance and angle constraints between points 1-2-5 ofthe pharmacophore of FIG. 4.

FIG. 13 shows the distance and angle constraints between points 1-2-3 ofthe pharmacophore of FIG. 4.

FIG. 14 shows the distance and angle constraints between points 2-3-5 ofthe pharmacophore of FIG. 4.

FIG. 15 shows the distance and angle constraints between points 2-4-5 ofthe pharmacophore of FIG. 4.

FIG. 16 shows the distance and angle constraints between points 1-3-5 ofthe pharmacophore of FIG. 4.

FIG. 17 shows the distance and angle constraints between points 2-3-4 ofthe pharmacophore of FIG. 4.

FIG. 18 shows the distance and angle constraints between points 1-3-4 ofthe pharmacophore of FIG. 4.

FIG. 19 shows the distance and angle constraints between points 3-4-6 ofthe pharmacophore of FIG. 4.

EXAMPLES

Here we demonstrate that Der p I, a major allergen of house dust mite(Dermatophagoides pteronyssinus), cleaves CD23 from the surface ofcultured human B cells (RPMI 8866 B cell line). The cleavage of thereceptor from the B cell surface was associated with a parallel increasein sCD23 in the culture supernatant. Labelled antibody experiments andprotease inhibition assays clearly demonstrate that Der p I is acysteine protease that directly cleaves a 25K fragment of CD23. Theproteolytic affect of Der p I has specificity for CD23, since none ofthe other B cell markers tested (CD20, HLA-DR, CD71 and CD49d) wereaffected. These data suggest that Der p I elicits IgE antibody responsesin 80% of patients suffering from dust mite allergy, by its ability toproteolytically release sCD23, and thereby upregulate IgE synthesis.

We have affinity purified Der p I from dust mite extract and tested itsability to proteolytically cleave CD23 expressed on cultured RPMI 8866 Bcells, using FITC labelled monoclonal anti-human CD23 (Bu38). The datashow that, in the presence of cysteine (5 mM), Der p I cleaves in adose-dependent manner membrane CD23, thereby releasing sCD23 into theculture supernatant (FIG. 1a and 1b). The proteolytic activity of Der pI was inhibited by E64 (a cysteine protease inhibitor), but not byalpha-1-antitrypsin (a serine protease inhibitor), thereby confirmingthe cysteine protease nature of Der p I (FIG. 1c). We have in factdemonstrated that Der p I completely cleaves alpha-1-antitrypsin (1:10molar ratio) to yield a degradation pattern (FIG. 1d) similar to thatgenerated by papain, a well characterised cysteine protease. A moredetailed description of FIG. 1 is as follows:

Cells were analysed on a FACScan (Becton Dickinson, Oxford, UK) with alinear fluorescence setting of 660 volts. The fluorescence (FL1) profileversus forward scatter (FSC) was used to monitor the cells, theamplification scale was altered according to the level of fluorescence.For each sample 4000 events were collected and then analysed using theflowMATE programme (DAKO, High Wycombe, UK). Data presented arerepresentative of 3 replicate experiments, each point in a to crepresents the mean of duplicate determinations, FIG. 1(a). Dose andcysteine dependency of CD23 cleavage by Der p I (for method ofpurification see FIG. 1(d) below). Der p I was pre-incubated (15 min at37° C.) with or without 5 mM cysteine and added to 2-3×10⁵ RPMI 8866cells in a total volume of 200 ml RPMI 1640+10 mM HEPES. The mixture wasthen incubated (1 h at 37° C.) and the cells, collected bycentrifugation, were washed in RPMI 1640+10 mM HEPES and incubated (30min at room temperature) with FITC conjugated anti-CD23 monoclonalantibody (Bu38, The Binding Site, Birmingham, UK). FIG. 1(b). Cleavageof membrane CD23 was associated with a parallel dose dependent releaseof sCD23 in the culture supernatant. The supernatant was collected fromcultured RPMI 8866 cells treated with Der p I (as described above) anddiluted 1/5 for sCD23 determination by ELISA (open circles) (The BindingSite, Birmingham, UK). In this ELISA there was no cross-reactivitybetween Der p I and sCD23. FIG. 1(c). Class specific inhibitor ofcysteine proteases prevent membrane CD23 cleavage by Der p I. E64(L-trans-epoxysuccinylleucylamido (4-guanidino) butane) (Sigma, Poole,UK) completely inhibits cleavage of CD23 by Der p I, whereas no suchinhibitory effect was demonstrable with alpha-1-antitrypsin, a naturallyoccurring human serine protease inhibitor. One hundred ml of 5 g/ml Derp I was pre-incubated (30 min at 37° C.) with 10 ml of either E64 oralpha-1-antitrypsin and then added to the RPMI 8866 cells (as describedabove). The arrows indicate level of CD23 expression in the absence(upper arrow) and presence (lower arrow) of Der p I. FIG. 1(d). Silverstain SDS-PAGE (12% gel) analysis of the Der p I preparation, humanalpha-1-antitrypsin and the effect of Der p I on alpha-1-antitrypsin.Der p I was purified by affinity chromatography using anti-Der p Iantibody (4Cl, Indoor Biotechnologies, Clwyd, UK). The purity of thepreparation was confirmed by N-terminal sequencing carried out on anautomatic amino acid sequencer (Applied Biosystems, Foster City, Calif.,USA). The sequence obtained (Thr-Asn-Ala-Cys-Ser-Ile-Asn-Gly-Asn-Ala, orTNACSINGNA SEQ ID No. 1) matches the published sequence of Der p I. Theactivity of the alpha-1-antitrypsin preparation was ascertained byactive site titration against bovine chymotrypsin (Dr. David Lomas,personal communication). The gel shows single bands for Der p I (lane 1)and alpha-1-antitrypsin (lane 2). Incubation (2 h at 37° C.) of Der p I(0.25 mg) with alpha-1-antitrypsin (5 mg), in a total volume of 10 ml,results in the cleavage of a large fragment (arrow) fromalpha-1-antitrypsin (lane 3). This pattern is in agreement with thatgenerated by papain. The mass standards are indicated on the left. Toinvestigate the enzymatic specificity of Der p I for CD23, we monitoredthe expression of other B cell markers following treatment with 2.5mg/ml (final concentration) of Der p I. At this Der p I concentration,which has been shown to give maximum cleavage of CD23 (FIG. 1a), therewas no significant loss of CD23, HLA-DR, CD71 and CD49d expressions(FIG. 2). A more detailed description of FIG. 2 is as follows:

RPMI 8866 cells were treated with 100 ml of 5 mg/ml Der p I and theexpression of membrane CD23 was monitored in parallel with other B cellsurface markers (CD20, HLA-DR, CD71 and CD49d). These markers weredetected using anti-CD20 (L27), anti-HLA-DR (L243) (Becton Dickinson,Oxford, UK), anti-CD71 (Ber-T9) (Dako, Buckinghamshire, UK) andanti-CD49d (HP2.1) (Immunotech, Westbrook, Me., USA) antibodiesrespectively. Paired results represent the expression of markers in theabsence (open bars) and presence (solid bars) of Der p I. Data presentedare representative of 3 replicate experiments, each point represents themean of duplicate determinations.

To gain insight as to the Der p I cleavage site on CD23, we onitored theproteolytic cleavage process using Bu38 and EBVCSI monoclonal anti-CD23antibodies, which are directed against the lectin domain and the stalkregion respectively. Thus, Bu38 detects all fragments down to 25 kDa,whereas EBVCSI recognises only fragments larger than 25 kDa (J. Gordon,personal communication). The results show that Der p I cleaves CD23 at asite close to the lectin domain, since EBVCS1 antibody was still capableof binding to the residual membrane bound portion of the receptor (FIG.3). However, at a Der p I concentration of greater than 1 mg/ml therealso appeared to be some cleavage of CD23 fragments larger than 25 kDa.Since the highest concentration of Der p I (2.5 g/ml) resulted incomplete loss of Bu38 binding and only partial loss of EBVCS1 binding,the preferred site of initial cleavage of CD23 by Der p I appears to beclose to the lectin domain. A more detailed description of FIG. 3 is asfollows:

RPMI 8866 cells were treated with Der p I, as described above, and theexpression of membrane CD23 was monitored using two monoclonal anti-CD23antibodies: Bu38 (recognises the lectin domain) and EBVCS1 (recognisesthe stalk region between 25 kDa fragments). Thus, Bu38 recognises theintact molecule and all soluble fragments, while EBVCS1 recognises theintact molecule and the residual membrane bound portion after cleavageof the 25 kDa fragment (sCD23) (J. Gordon, personal communication). Theexperiment demonstrates that Der p I, at concentrations of up to 1mg/ml, preferentially releases a 25 kDa fragment of CD23. Data presentedare representatuve of 3 replicate experiments, each point represents themean of duplicate determinations.

The preferred cleavage site of CD23 giving rise to the 25 kD fragmenthas been identified by us as detailed above.

Soluble CD23 is one of the signals known to induce IgE producing B cellsto become plasma cells which are required for IgE production. Thereforethe nature of the proteases that cleave CD23 in vivo is of considerableinterest. Although it has been suggested that CD23 has autoproteolyticactivity, we were previously unaware of what proteases cleave membraneCD23. We have demonstrated that Der p I, an exogenous cysteine protease,fulfils this function. Der p I elicits IgE antibody responses in 80% ofpatients suffering from dust mite allergy, and there is in vivo evidencethat such patients have high circulating levels of sCD23. Thisubiquitous inhaled allergen is clearly highly immunogenic, and webelieve its immunogenicity may be due in part to its enzymatic activity.It has indeed been demonstrated that the allergenicity of papain, acysteine protease showing sequence homology with Der pI, is highlyrelated to its enzymatic activity.

The demonstration that Der p I proteolytically cleaves membrane CD23raises the question of the role of IgE in the allergic process. Firstly,IgE specific to Der p I could target Der p I to B lymphocytes and otherCD23 bearing cells (e.g. eosinophils), thereby helping to build a highconcentration of this allergen on the cell surface. Secondly, thebinding of IgE to CD23 may protect the receptor from proteolytic attackby Der p I.

Purification of Der p I Protein

Crude mite extract (˜100 mg, SmithKline-Beecham) was dissolved in 5 mlPhosphate Buffered Saline (PBS; 50 mM potassium phosphate; pH 7.4containing 150 mM NaCl). Der p I was purified by affinity columnchromatography using 4 C1 antibody (Indoor Biotechnology, Deeside, U.K.)immobilised onto CNBr activated Sepharose 4B (Pharmacia, Milton Keynes,U.K.). The crude preparation was mixed with -2 ml of the affinity resinfor 2 h at 4° C. and then washed with 2-3 volumes of PBS. Elution ofbound protein was carried out using 5 mM glycine containing 50% (v/v)ethylene glycol. Fractions (1-2 ml) were collected and neutralised with0.8 ml of 0.2 M sodium phosphate buffer, pH 7.0. The fractions werepooled and dialysed overnight against 4 L PBS followed by a seconddialysis against 2 L PBS for 2-3 h. The total protein was concentratedas required by ultrafiltration (MacroSep; Flowgen, U.K.).

This yielded protein of greater than 95% purity as judged by denaturingpolyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate, C4 reverse phase high performance liquid chromatography(RP-HPLC) and high pressure size exclusion chromatography (HP-SEC) andno other contaiminating protease activity could be detected.

Inhibitors of Der p I

Using the purified Der p I it was surprisingly found that inhibitors tothe enzyme could be made. These inhibitors are of the general formula##STR2## where X, Y, and Z may be N or CH.

R₁ is a blocking group for the N-terminal amino acid nitrogen (T.Greene. Protective Groups In Organic Synthesis). R₂, R₃, and R₄ areside-chains on X, Y, and Z.

W is a group that reactions irreversibly with an active cysteine thiolof Der p I.

Where X and Y are CH, stereochemistry is exclusively of the "S"configuration, providing L -alpha-amino acid residues. Where Z is CH,the configuration may be "R" or "S" dependent upon W, but the chiralcentre is derived stereospecifically with retention of configurationfrom the L -alpha-amino acid precursor. Where X, Y or Z are nitrogen,the residue is a peptidomimetic, an "azapeptide".

Preferably, R₁ represents an optionally substituted hydrophobic aryl orheteroaryl group optionally connected through a heteroatom (O, S, N, P)to the carbonyl. When connected through N or P the heteroatom may bemono or diaryl or mono or diheteroaryl substituted.

Alternatively, R₁ represents a hydrophobic aliphatic group of 3 carbonsor more, linear or branched optionally connected through a heteroatom(O, S, N, P) to the carbonyl. When connected through N or P, theheteroatom may be mono or di-substituted.

These compounds can also be optionally substituted aryl for exampleoptionally substituted phenyl, naphthyl or unsubstituted 2-naphthyl or9-anthracyl. Additionally, optionally substituted phenyl may beuonsubstituted phenyl or phenyl having 1 to 5 fluoro substituents orphenyl having 1 to 3 substituents where the substituents areindependently selected from the group which comprises lower alkyl, loweralkoxy, nitro, halo, acetyl, benzoyl, hydroxyl, amino, methylamino,--COOH, --CONH₂, --COOR², and NHCOR² wherein R² is lower alkyl.

Optionally substituted 1-naphthyl includes unsubstituted 1-naphthyl and1-naphthyl substituted at the 2-position with lower alkyl, lower alkoxyor trifluoromethyl.

Optionally substituted heteroaryl includes optionally substituted, 5 or6 membered aromatic group containing 1 to 4 heteroatoms chosen from O,S, N, a 1 or 2-naphthyl or a 9-anthracyl group which may contain 1 to 4heteroatoms chosen from O, S, and N.

Most preferably R₁ represents pkhenyl, diphenyl amino radical,9-xanthenyl, piperonyl, phenyl amino radical, tert-butoxy, CF₃ -phenyl,a mono or disubstituted phenyl where the substituent is a lower alkylC1-3, lower alkoxy C1-3, mono 2 or 3 amino or carboxy substitutedphenyl, These criteria will also apply for diphenylamino radical and9-xanthenyl. In addition, straight chain or branched aliphatics such aspivolyl, n-butyl and variants thereof upto C8.

Preferably R₂ represents a hydrophobic side-chain as found bonded to theC-alpha of commercially available amino acids. Hydrophobic refers tostraight or branched chain alkyl (Methyl such as Ala); cyclohexylmethyl;2-methylpropyl i.e. Leu; n-butyl i.e. Norleucine; 1-methylethyl i.e.Val; 1-methylpropyl i.e. Ile; 3-methylbutyl, i.e. homoleucine; ethyli.e. Abu.

Alternatively, the hydrophobic chain may contain a heteroatom such as N,O, S such as 2-methylthioethyl (methionine), 4-aminobutyl i.e. Lys; orethyl-2-carboamide i.e. Gln.

Alternatively, the hydrophobic chain may be a phenylmethyl radicaloptionally containing a nitrogen atom or be substituted on the phenylring with --OH, alkoxy, phenyl, or alkyl at C1-3.

Most preferably R₂ represents biphenylmethyl, 1-methylethyl i.e. valine;methyl i.e. alanine; or cyclohexylmethyl i.e. cyclohexylalanine.

Preferably R₃ represents a C1 alkyl group optionally substituted with aheteroatom, O, or F. Alternatively, R₃ may be 4-aminobutyl i.e. Lys;ethyl-2-carboxamide i.e. Gln; 2-(methylthiooxy) ethyl i.e. Met(O).

Most preferably, R₃ represents methyl i.e. alanine.

Preferably, R₄ represents a hydrophobic side-chain defined and withresidues as described for R₂. In addition, 2-hydroxyethyl, i.e. Thr; or2-fluoroethyl.

Most preferably R₄ represents 3-methylbutyl i.e. homoleu;cyclohexylmethyl i.e. cha; 2-methylpropyl i.e. leucine; or n-butyl i.e.norleucine.

Preferably W is selected from the group which comprises: ##STR3##

Preferably E is selected from the group which comprises:

i) OAr or SAr ##STR4## iii) heteroaryl iv) halogen ##STR5##

Preferably R is selected from the group which comprises alkyl and Ar.

Preferably Ar is selected from the group which comprises optionallysubstituted aryl of heteroaryl.

Preferably Y is selected from the group which comprises esters,sulphones, carboxylates, amides, phosphonates, ketones, sulfonates,nitriles, sulphonamides and nitro compounds.

DEFINITIONS

Optionally substituted aryl is preferably optionally substituted phenyl,benzyl or naphthyl. Optionally substituted phenyl is preferablyunsubstituted phenyl or phenyl having 1 to 5 fluoro substituents orphenyl having 1 to 3 substituents where the substituents areindependently selected form the group comprises lower alkyl, loweralkoxy, nitro, halo, acetyl, benzoyl, hydroxy, amino, methylamino,dimethylamino, diethylamino, methylthio, cyano, trifluoromethyl,phenylsulfonamidecarbonyl (--CONHSO₂ C₆ H₅), --COOH, --CONH, --COOR,NHCOR wherein ₂ R is lower alkyl and2,3,5,6,-tetramethyl-4-carboxy-phenyl (--C₆ H₅ (CH₃)₄ --COOH).

Optionally substituted 1-naphthyl includes unsubstituted 1-naphthyl and1-naphthyl substituted at the 2-position with lower alkyl, lower alkoxy,or trifluoromethyl.

Halogen is preferably bromo, chloro or fluoro.

Alkyl is preferably a branched or unbranched, saturated aliphatichydrocarbon radical, having the number of carbon atoms specified, or ifno number is specified, having up to 8 carbon atoms. The prefix "alk--"is also indicative of a radical having up to 8 carbon atoms in the alkylportion of that radical, unless otherwise specified. Examples of alkylradicals include methyl, ethyl, n-propyl, iospropyl, n-butyl,tert-butyl, n-pentyl, n-hexyl, and the like. The terms "lower alkyl" and"alkyl of 1 to 4 carbon atoms" are, within the context of thisspecification, synonymous and used interchangeably.

Optional or optionally indicates that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, "optionally substituted phenyl" means that thephenyl radical may or may not be substituted and that the descriptionincludes both unsubstituted phenyl radicals and phenyl radicals whereinthere is substitution.

These inhibitors were exemplified by the following examples which wereproduced as detailed.

Synthesis of Der p I Inhibitors

Potential inhibitors for Der p I were synthesised according to thegeneral methods described below. Following synthesis the compounds weresubjected to electrospray or MALDI-TOF mass spectrometry (MS) and theresults are indicated.

Compound 1: N-Benzoyl-L-valyl-L-alanyl-L-norleucine

Solid phase benzoylated peptide synthesis.

Resin Loading (Step 1)

2-Chlorotritylchloride resin (4.9 g, 1.05 mmol/g, Novabiochem) wasswelled in dichloromethane (40 ml) and a suspension of Fmoc-L-norleucineadded and stirred for 5 minutes. A solution of diisopropylethylamine inDCM (10 ml, 57 mmol in 30 ml) was added over 5 minutes and the resultingmixture stirred at room temperature for 2 hours. Methanol (5 ml) addedand reaction mixture stirred for a further 10 minutes before resinfiltered and washed with 3×DCM, 2×DMF, 2×2-propanol, 2×DMF,2×2-propanol, methanol, 2×ether and dried under vacuum for 24 hours.

Amino Acid Deprotection (Step 2)

Fmoc-L-norleucine loaded resin was deprotected by treatment with 20%piperidine in DMF over 4 hours. The swollen resin was filtered, washedwith 5×DMF, 2×ether and dried under vacuum for 24 hours.

Peptide Chain Extension (Step 3)

L-Norleucine loaded resin (5 mmol) was added to a solution ofFmoc-L-alanine (6.23 g, 20 mmol), hydroxybenzotriazole (3.0 g, 20 mmol).2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (7.59 g, 20 mmol) and diisopropylethylamine (6.97ml, 40 mmol) in DMF (20 ml) and allowed to swell over 4 hours with mildagitation. Resin was filtered and washed with 4×DMF, 2×ether and driedunder vacuum overnight. Steps (2) and (3) were carried out repetitivelywith Fmoc-L-alanine and Fmoc-L-valine to afford resin bound tripeptideH-L-valyl-L-alanyl-L-norleucine.

Peptide Chain Benzoylation (Step 4)

L-Valyl-L-alanyl-L-norleucine loaded resin (1 g, approx. 1 mmol) wasadded to a solution of benzoic acid (0.488 g, 4 mmol),hydroxybenzotriazole (0.6 g, 4 mmol),2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (1.52 g, 4 mmol) and diisopropylethylamine (1.40 ml,8 mmol) in DMF (5 ml) and allowed to sell over 6 hours with mildagitation. Resin was filtered and washed with 4×DMF, 2×ether and driedunder vacuum overnight.

Resin Cleavage (Step 5)

N-Benzoyl-L-valyl-L-alanyl-L-norleucine loaded resin (1.0 g, appr. 1mmol) was treated with a 1% solution of trifluoroacetic acid indichloromethane (20 ml) containing triethylsilane (320 μl, 2 mmol) for 1hour. Resin was removed by filtration and washed with dichloromethane(3×10 ml). Organic layer was collected, evaporated and triturated withether to afford N-benzoyl-L-valyl-L-alanyl-L-norleucine (285 mg).

Electrospray MS m/z 407 [MH⁺ ].

Compound 2 N-Benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone

N-Benzoyl-L-valyl-L-alanyl-L-norleucine (140 mg, 0.34 mmol) wassuspended in dry THF (3 ml) and dry DMF was added dropwise to affordhomogeneity. The reaction mixture as cooled to -10° C. andisobutylchloroformate (129 μl, 1.0 mmol) and N-methylmorpholine (109 μl,1.0 mmol) added with stirring under Argon. The mixture was stirred for30 minutes before a solution of diazomethane in ether (5 ml, approx. 2mmol) was added. The reaction mixture was allowed to warm to roomtemperature over 1 hour before a 1:1 solution of acetic acid and 50% HBr(1 ml, 3.0 mmol HBr) was added dropwise and stirred for 15 minutes. Theorganic phase was diluted with ethylacetate (40 ml), washed with water(10 ml), brine (10 ml) and sat. bicarbonate (2×10 ml), dried over MgSO₄solvent removed under vacuum. This afforded an off white solid (152 mg)which could be further purified as required by preparative HPLC.Electrospray MS m/z 482 [MH⁺ ] and 484 [MH⁺ ].

Compound 3 N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,6-bis(trifluoromethyl)benzoyloxy methyl ketone

A mixture of potassium fluoride (0.1 mmol, 6 mg) and2,6-bis(trifluoromethyl)benzoic acid (0.066 mmol, 17 mg) in dry DMF (500μl) was stirred over molecular sieves at room temperature for 5 minutes.A solution of N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone(0.033 mmol), 16 mg) in dry DMF (500 μl) was added and the reactionmixture stirred for 1 hour. The reaction mixture was passed through ashort silica plug and washed with 5% methanol in dichloromethane.Solvent was removed under vacuum and the residue purified using prep.HPLC. Freeze drying afforded (6.4 mg) as a white lyophilisate.Electrospray MS m/z 660 [MH⁺ ]. ##STR6##

Similarly the following compounds were prepared.

Compound 4 N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,6-dimethylbenzoyloxy methyl ketone

(Electrospray MS m/z 552 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and2,6-dimethylbenzoic acid.

Compound 5 N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2-hydroxybenzoyloxymethyl ketone

(Electrospray MS m/z 540 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and2-hydroxybenzoic acid.

Compound 6 N-Benzoyl-L-valyl-L-alanyl-L-norleucine2,6-dichlorobenzoyloxymethyl ketone

(Electrospray MS m/z 592 [MH⁺ ] and 594 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and2,6-dimethylbenzoic acid.

Compound 7 N-Benzoyl-L-valyl-L-alanyl-L-norleucine benzoyloxymethylketone

(Electrospray MS m/z 524 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and benzoicacid.

Compound 8 N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,3,4,5,6-pentafluorobenzoyloxy methyl ketone

(Electrospray MS m/z 614 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and2,3,4,5,6-pentafluorobenzoic acid.

Compound 9 N-Benzoyl-L-valyl-L-alanyl-L-norleucine1,1-dimethylpropyloxymethyl ketone

(Electrospray MS m/z 504 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and1,1-dimethylpropanoicacid.

Compound 10 N-Benzoyl-L-valyl-L-alanyl-L-norleucineN-(-benzyloxycarbonyl)-D-serinyl-(O-tert-butyl)oxymethyl ketone

(Electrospray MS m/z 697 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone andN-benzyloxycarbonyl-D-serinyl-O-tert-butylether.

Compound 11 N-Benzoyl-L-valyl-L-alanyl-L-norleucineN(-benzyloxycarbonyl)-D-serinyloxy methyl ketone

(Electrospray MS m/z 641 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethly ketone andN-benzyloxycarbonyl-D-serine.

Compound 12 N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2-furanoxy methylketone

(Electrospray MS m/z 514 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 2-furancarboxylic acid.

Compound 13 N-Benzoyl-L-valyl-L-alanyl-L-norleucine2,6-dichlorophenylacyloxy methyl ketone

(Electrospray MS m/z 606 [MH⁺ ], 608 [MH⁺ ]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and2,6-dichlorophenylacetic acid.

Standard preparative HPLC conditions were used to analyse thesecompounds thus C4 preparative HPLC system (Vydac, 22×250 mm) eluting at10 ml per minute a gradient of 5-95% (90% acetonitrile (0.1% TFA)) over30 minutes.

Compound 14 N-Benzoyl amino-L-valyl-L-alanyl-L-norleucyl-hydroxamic acid

To a suspension of Bz-Val-Ala-norLeu-OH (50 mg, 0.12 mmol) in THF (5 ml)in a plastic reaction vessel was added diazomethane (0.3 mmol) in ether1.5 ml. Gas evolution was observed and to the resulting clear solutionwas added acetic acid (0.05 ml) and the solution was evaporated todryness. The residue was dissolved in methanol (2 ml) and hydroxylamine(2 mmol) in methanol (2 ml) was added and the solution stirred for 5hours at room temperature. The solution was concentrated water added (2ml), and the resulting solid was filtered and dried to yield 33 mg, 65%.

Compound 15 N-(Benzoyl amino-L-valyl-L-alanyl-L-norleucyl)-O-benzoylhydroxamate

To a solution of Bz-Val-Ala-norLeu-NHOH (10 mg, 0.022 mmol) in drypyridine at -10° C. was added benzoyl chloride (0.004 ml, 0.03 mmol) andstirred for 2 hours. The solution was evaporated and purified accordingto the method described in the preparation ofEthyl-(S)-(E)-3-((N-benzoyl valyl alanyl)amino-6-methyl-hept-2-enoate,collecting the peak elution at 25-27 min. and lyophilised to yield 0.5mg, 5%.

Electrospray MS m/z 525 [MH⁺ ].

Compound 16N-(N-benzoyl-L-valyl-L-alanyl-L-norleucyl)-O-2,6-dimethyl-benzoylhydroxamate

To a solution of 2,6-dimethylbenzoic acid (4 mg, 0.024 mmol) in dry DMF(1 ml) cooled to 0° C. was added 1-hydroxy-7-azabenzotriazole (3.2 mg,0.023 mmol), O-7-azabenzotriazole-1-yl-1,1,3,3-tetramethyl uroniumhexafluorophosphate (9 mg, 0.023 mmol) and N-methylmorpholine (0.008 ml,0.07 mmol) and the solution was stirred for 5 minutes. The hydroxamicacid Bz-Val-Ala-norLeu-NHOH (10 mg, 0.02 mmol) was added and thereaction stirred overnight. The solution was evaporated and purifiedaccording to the method described in the preparationEthyl-(S)-(E)-3-((N-benzoyl valyl alanyl) amino-6-methyl-hept-2-enoatecollecting the peak eluting at 26-28 min. and lyophilised to yield 0.9mg, 6%.

Electrospray MS m/z 553 [M⁺ +H], 575 [M⁺ Na]

Compound 17 Preparation of N,O-dimethyl(tert-butoxycarbonylamino-L-leucyl)hydroxylamine

A solution of Boc-Leu-OH.H₂ O (80.3 mmol) and N-methyl morpholine (88mmol) in THF (35 ml) was added to a pre cooled solution of isobutylchloroformate (88 mmol) in THF (65 ml) under nitrogen at between -10 and-15° C. over 40 minutes. The reaction was stirred at -10° C. for 1 hourafter which time N-methyl morpholine (88 mmol) was added followed byN,O-Dimethylhydroxylamine hydrochloride (88 mmol) portion wise between-10 and 0° C. The reaction was then stirred at -10° C. for 1 hour andthen allowed to warm up to room temperature over night. The THF was thenremoved under vacuum and water (50 ml) and ethylacetate (200 ml) added.The organic layer was then washed with 0.1 M citric acid solution (4×50ml), then saturated sodium bicarbonate (4×50 ml), dried over magnesiumsulphate and then concentrated under vacuum to give the product.

Compound 18 Preparation of N,O-dimethyl(amino-L-leucyl) hydroxylamine

Hydrogen chloride in dioxane (4M, 75 mL) was added to Boc-Leu-N(OMe)Me(33 mmol) with cooling and then stirred at room temperature for 1 hour.The solution was then concentrated under vacuum. Diethyl ether (100 ml)added and concentrated down to dryness three times to give the product.

Compound 19 Preparation of N,O-dimethyl (tert-butoxycarbonylamino-L-alanyl-L-leucyl) hydroxylamine

A solution of Boc-Ala-OH (46 mmol) and N-methyl morpholine (46 mmol) inTHF (20 ml) was added to a pre cooled solution of isobutyl chloroformate(46 mmol) in THF (30 ml) under nitrogen at between -10 and -15° C. over30 minutes. The reaction was stirred at -10° C. for 1 hour after whichtime a solution of N-methyl morpholine (46 mmol) andHCl.H2N-Leu-N(OMe)Me (41.8 mmol) in 1,4-dioxane (20 ml) was added dropwise slowly. The reaction was left for 1 hour at -10° C. and thenallowed to warm up to room temperature. After concentrating the solutionunder high vacuum, water (50 ml) and ethylacetate (200 ml) was added.The organic layer was then washed with 0.1 M citric acid solution (4×50ml), then saturated sodium bicarbonate (4×50 ml), dried over magnesiumsulphate and then concentrated under vacuum to give the product.

Compound 20 Preparation ofN,O-dimethyl(amino-L-alanyl-L-leucyl)hydroxylamine

Hydrogen chloride in dioxane (4M, 80 mL) was added toBoc-Ala-Leu-N(OMe)Me (33 mmol) with cooling and then stirred at roomtemperature for 1.5 hour. The solution was then concentrated undervacuum. Diethyl ether (100 ml) was then added and concentrated down todryness three times to give the product.

Compound 21 Preparation of N,O-dimethyl(tert-butoxycarbonylamino-L-valyl-L-alanyl-L-leucyl)hydroxylamine

A solution of Boc-Val-OH (46 mmol) and N-methyl morpholine (46 mmol) inTHF (20 ml) was added to a pre cooled solution of isobutyl chloroformate(46 mmol) in THF (30 ml) under nitrogen at between -10 and -15° C. over30 minutes. The reaction was stirred at -10° C. for 1 hour after whichtime a solution of N-methyl morpholine (46 mmol) andHCl.H2N-Ala-Leu-N(OMe)Me (41.8 mmol) in 1,4-dioxane (30 ml) was addeddrop wise slowly. The reaction was left for 1 hour at -10° C. and thenallowed to warm up to room temperature. After concentrating the solutionunder high vacuum, water (50 ml) and ethylacetate (200 ml) was added.The organic layer was then washed with 0.1 M citric acid solution (3×50ml), then saturated sodium bicarbonate (3×50 ml), dried over magnesiumsulphate and then concentrated under vacuum to give the product.

Electrospray MS m/z 445 [MH⁺ ]

Compound 22 Preparation of tert-butoxycarbonylamino-L-valyl-L-alanyl-L-leucyl aldehyde

A solution of lithium aluminium hydride (4.5 mmol)in THF (24.5 mL) wascooled to between -15 and -10° C. Boc-Val-Ala-Leu-N(OMe)Me (2.2 mmol) inTHF (10 mL) was then added very slowly to maintain the low temperature.After 40 minutes ethyl acetate (10 mL) was added slowly at -15° C. andthen left for 10 minutes. Water (2 mL) was then added very slowly, againat -15° C. and the reaction then allowed to warm up to room temperature.Citric acid solution (100 mL, 0.5M) was then added and the productextracted into ethyl acetate. The ethyl acetate layer was washed with100 ml saturated sodium bicarbonate solution, followed by 100 ml waterand then dried over magnesium sulphate. The solution was thenconcentrated to give the product which was subsequently used crude.

Electrospray MS m/z 386 [MH⁺ ]

Compound 23 Ethyl-(S)-(E)-3-((tert-butoxycarbonylamino-L-valyl-L-alanyl) amino-6-methyl-hept-2-enoate

To a suspension of sodium hydride (46 mg, 1.9 mmol) in anhydrous THF (4ml) cooled to 0° C. was added a solution of triethylphosphonoacetate(420 mg, 1.9 mmol) in THF (2 ml) dropwise over 5 minutes and the mixturestirred until gas evolution ceased. The solution was added dropwise to asolution of Boc-Val-Ala-Leucyl aldehyde (600 mg, 1.56 mmol) in dry THFcooled to -10° C. The reaction mixture was stirred for 1 hour andsaturated ammonium chloride (10 ml) was added. A white solidprecipitated which was removed by filtration and the filtrate waspartitioned between ethyl acetate and water. The organic layer was driedwith magnesium sulphate and evaporated to give an oil which wascrystallised from acetonitrile water to yield the title compound, 640mg, 91%.

Electrospray MS m/z 456 [M⁺ +H], 356 [(M⁺ -^(t) BOC)+1]

Compound 24 (S)-(E)-3-((tert-butoxycarbonyl amino-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoic acid

Ethyl-(S)-(E)-3-((tert-butoxy carbonyl amino valyl alanyl)amino-6-methyl-hept-2-enoate (455 mg, 1 mmol) was dissolved in dioxane(10 ml) was water added followed by lithium hydroxide (126 mg, 3 mmol).The solution was stirred for 3 hours and 1M HCl aq was added until thepH reached neutrality. The dioxane was removed by rotary evaporation andthe pH adjusted to 4 with 1M HCl aq. The title compound precipitated,filtered and washed with water to yield 420 mg, 98%.

Electrospray MS m/z 428 [M⁺ +H]

Compound 25 1,1,1-Trifluoroethyl-(S)-(E)-3-((tert-butoxycarbonylamino-L-valyl-L-alanyl) amino-6-methyl-hept-2-enoate

The acid (Boc-Val-Ala-Leu-OH) (50 mg, 0.117 mmol) anddimethylaminopyridine (29 mg, 0.24 mmol) was dissolved in drydichloromethane (1 ml) and cooled to 0° C. Water soluble carbodiimidehydrochloride salt (26 mg, 0.13 mmol) in 0.5 ml dichloromethane wasadded and the solution stirred for 5 minutes, 1,1,1-Trifluoroethanol(0.017 ml, 0.23 mmol) in 0.5 ml dichloromethane was added and thereaction was allowed to warm to room temperature after 1 hour and thereaction mixture stirred overnight. The reaction mixture was washed 2×2ml 0.5M citric acid solution, 1×2 ml water, 1×2 ml saturated sodiumbicarbonate solution, 1×2 ml water, dried with magnesium sulphate andevaporated to dryness to give the title compound

Electrospray MS m/z 510 [M⁺ +H], 410 [(M⁺ -^(t) BOC)+1], 454 [(M⁺ -^(t)Bu)+1]

Compound 26 Ethyl-(S)-(E)-3-((N-benzoyl-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoate

The Ethyl-(S)-(E)-3-((tert-butoxycarbonyl amino valyl alanyl)amino-6-methyl-hept-2-enoate (16.6 mg, 0.036 mmol) was dissolved in 4.0MHCl in dioxane (2 ml) stirred at room temperature for 30 minutes andevaporated to dryness. The residue was dissolved in DMF (0.5 ml) andN-methylmorpholine (7.36 mg, 0.073 mmol) added followed by benzoylchloride (5.4 mg, 0.038 mmol) in DMF 0.5 ml. The reaction stirred for 2hours, diluted with 0.1% trifluoroacetic acid solution (4 ml) andacetonitrile (2 ml) and injected onto a C4 preparative HPLC system(22×250 mm) eluting at 10 ml per minute, monitoring at 215 nm and agradient of 10-90% system B over 25 minutes and holding at 90% for 15minutes. System A=0.1% TFA in water, system B=90% acetonitrile, 10%system A. The peak eluting at 26-28 minutes was collected andlyophilised to a white solid, yield 4.5 mg, 27%.

Electrospray MS m/z 460 [M⁺ +H]

In an identical manner to the above, the following compounds wereprepared:

Compound 27Ethyl-(S)-(E)-3-((2-trifluoromethyl-N-benzoyl-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoate

A yield of 3.7 mg, at 22% was obtained. Electrospray MS m/z 528 [M⁺ +H].

Compound 28 Ethyl-(S)-(E)-3-((Piperonyloyl amino-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoate

yield 3.8 mg, 23%.

Electrospray MS m/z 504 [M⁺ +H].

Compound 29 Ethyl-(S)-(E)-3-((Phenyl carbamoyl amino-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoate

As per method above, except that phenyl isocyanate was used in place ofan acid chloride, yield 1.5 mg, 10%.

Electrospray MS m/z 475 [M⁺ +H].

Compound 30 Ethyl-(S)-(E)-3-((Diphenyl carbamoyl amino-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoate

yield 2.3 mg, 13%.

Electrospray MS m/z 551 [M⁺ +H].

Compound 31 Ethyl-(S)-(E)-3-((Naphthoyl amino-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoate

yield 1 mg, 6%.

Electrospray MS m/z 510 [M⁺ +H].

Compound 32 Ethyl-(S)-(E)-3-((Quinazoloyl amino-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoate

yield 1.5 mg, 9%.

Electrospray MS m/z 512 [M⁺ +H].

Compound 33 Ethyl-(S)-(E)-3-((Morpholinoyl amino-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoate

yield 2.9 mg, 19%.

Electrospray MS m/z 469 [M⁺ +H].

Compound 34 Ethyl-(S)-(E)-3-((Xanthene-9-oyl amino-L-valyl-L-alanyl)amino-6-methyl-hept-2-enoate

As per method above, except that xanthane-9-carboxylic acid (8.1 mg,0.036 mmol) was used in place of the acid chloride. Coupling of thisacid was effected using2-(1H-benzotriazole-1-yl)-1,1,3,3-teramethyluronium hexafluorophosphate(13.6 mg, 0.036 mmol), as activator and 1-hydroxybenzotriazole (5.5 mg,0.036 mmol) as catalyst in the presence of N-methylmorpholine (10.8 mg,0.108 mmol).

Yield 1.7 mg, 9%.

Electrospray MS m/z 564 [M⁺ +H].

Compound 35 Diethyl Phenylsulfonylmethylphosphonate

(Adapted from I. Shahak, J. Almog, Synthesis 145 (1970).) Thecommercially available diethyl phenylthiomethylphosphonate (1.0 ml, 4.1mmol) was dissolved in dichloromethane (10 ml). Sulphuric acid (10 ml,25%) was added and the mixture cooled in ice. Solid Potassiumpermanganate was then added portionwise (3×0.5 g) with stirring afterwhich time the reaction appeared to be complete. Solid sodiummetabisulfite was added slowly until the mixture turned colourless. Thiswas then extracted with ethyl acetate (×3) and the combined organicwashings washed with saturated sodium bicarbonate solution followed bybrine before drying over sodium sulphate. The volatiles were thenremoved in vacuo. The residue was purified by flash chromatography onsilica eluting initially with ethyl acetate/hexane 8/2 followed by pureethyl acetate. In this way the desired product, diethylphenylsulfonylmethylphosphonate (1.0 g, quant) was obtained as acolourless solid.

MS (MALDI-TOF): required (M⁺ (C₁₁ H₁₇ O₅ PS)+1)=292; obtained (M⁺+1)=292 ##STR7##

Compound 36(S)-(E)-3-((tert-butoxycarbonylamino-L-valyl)-L-alanyl)amino-1-phenylsulfonyl-5-methyl-1-hexene

Diethyl phenylsulfonylmethylphosphonate (38 mg, 129 mmol) was dissolvedin dry THF (10 ml) and then cooled to 0° C. under an atmosphere ofnitrogen. Sodium hydride (8 mg of 60% dispersion in oil, 200 mmol) wasadded and the mixture stirred for 15 mins (effervescence). The aldehyde^(t) Boc-Val-Ala-Leucyl aldehyde (50 mg, 129 mmol) was then added to theresulting solution and the mixture was stirred for 60 mins. The reactionwas quenched by addition of dilute hydrochloric acid (0.1 M), followedby extraction with ethyl acetate (×3). The separated organic phase wassequentially washed with saturated sodium bicarbonate solution and brinebefore drying over sodium sulphate. The volatiles were removed in vacuo.The residue was purified by flash chromatography on silica eluting withethyl acetate/hexane 4/6. An unidentified by-product was eluted first(12 mg) followed by the desired product(S)-(E)-3-(tert-butoxycarbonyl-amino-L-valyl-L-alanyl)amino-phenylsulfonyl-5-methyl-1-hexene(22 mg, 32%) as a solid.

Electrospray MS m/z 546 [M⁺ +Na], 424 [(M-^(t) Boc)+1] ##STR8##

Compound 37 Diethyl Methylsulfonylmethylphosphonate

The commercially available Diethyl methylthiomethylphosphonate wasconverted to the title compound using the method of I. Shahak and J.Almog, Synthesis 171 (1969). ##STR9##

Compound 38(S)-(E)-3-((tert-butoxycarbonylamino-L-valyl)-L-alanyl)amino-1-methylsulfonyl-5-methyl-1-hexene

Diethyl methylsulfonylmethylphosphonate (30 mg, 130 mmol) was dissolvedin dry THF (5 ml) and then cooled to 0° C. under an atmosphere ofnitrogen. Sodium hydride (7 mg of 60% dispersion in oil, 175 mmol) wasadded and the mixture stirred for 15 mins (effervescence). The aldehyde^(t) Boc-Val-Ala-Leucyl aldehyde (50 mg, 129 mmol) was then added to theresulting solution and the mixture then stirred for 60 mins. Thereaction was quenched by addition of dilute hydrochloric acid (0.1 M),followed by extraction with ethyl acetate(×3). The separated organicphase was sequentially washed with saturated sodium bicarbonate solutionand brine before drying over sodium sulphate. The volatiles were thenremoved in vacuo. The residue was purified by flash chromatography onsilica eluting with ethyl acetate/hexane 8/2. An unidentified by-productwas eluted first (4 mg) followed by the desired product(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-methylsulfonyl-5-methyl-1-hexane(24 mg, 40%) as a solid.

Electrospray MS m/z 484 [M⁺ +Na], 362 [(M-^(t) Boc)+1] ##STR10##

Compound 39 Ethyl Diethylphosphorylmethylsulfonate

Prepared in accordance with procedure B in L. Ghosez et. al. Tetrahedron43 5125 (1987).

Electrospray MS m/z 261 [M⁺ +H], 283 [M⁺ +Na]. ##STR11##

Compound 40 Ethyl(S)-(E)-3-((tert-butoxycarbonylamino-L-valyl)-L-alanyl)amino-5-methylhexenylsulfonate.

Ethyl diethylphosphorylmethanesulfonate (36 ml, ˜138 mmol) was dissolvedin dry THF (5 ml) and then cooled to 0° C. under an atmosphere ofnitrogen. Sodium hydride (8 mg of 60% dispersion in oil, 200 mmol) wasadded and the mixture stirred for 15 mins (effervescence). The aldehyde^(t) Boc-Val-Ala-Leucyl aldehyde (50 mg, 129 mmol) was added to theresulting solution and the mixture stirred for 30 mins. The reaction wasquenched by addition of dilute hydrochloric acid (0.1 M), followed byextraction with ethyl acetate (×3). The separated organic phase wassequentially washed with sodium bicarbonate solution and brine beforedrying over sodium sulphate. The volatiles were then removed in vacuo.The residue was purified by flash chromatography on silica eluting withethyl acetate/hexane 1/1. The desired product,Diethyl(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-5-methylhexenylsulfonate, (22 mg, 35%) was obtained as a solid.

electrospray MS m/z 492 [M⁺ +1], 392 [(M⁺ -^(t) Boc)+1] ##STR12##

Determination of Kinetic Constant For Der-p I Substrates

All Der-p I enzyme assays were routinely carried out in 50 mM potassiumphosphate; pH 8.25 containing 1 mM ethylenediaminetetraaceticacid (EDTA)and 1 mM dithiothreitol (DTT). Product formation was monitored withrespect to time by measuring the increase in fluorescence emission at420 nm and exciting at 320 nm. All assays were carried out at 25° C.Stock solutions of the various substrates and/or inhibitors were made upin 100% dimethylsulphoxide (DMSO).

The kinetic constants (K_(M) and k_(cat)) were calculated from theinitial velocities of the enzymatic reaction at various substrateconcentrations. These data were fitted, by regression analysis, to theMichaelis-Menten equation and the kinetic constants obtained.

Inactivation Kinetics ##STR13##

The reaction of enzyme and inhibitor is comprised of two steps. Thefirst is binding of enzyme and inhibitor to produce the enzyme inhibitorcomplex (E.I). This step is assumed to be rapid and reversible, relativeto the other steps, and no chemical reaction occurs. In this case k₁ isthe second order rate constant for the formation of the E.I complex andk₋₁ is the first order rate constant for the breakdown of the E.I. Thesecond step in the process, occurring at a rate k₂, is the formation ofthe enzyme-inhibitor covalent complex (EI) resulting in irreversibleinactivation of the enzyme.

The practice of inactivation kinetics of enzymes have been described bytwo standard accepted methods (Schemes 1 and 2). The first (Scheme 1) isthe dilution method described by Kitz, C. G. and Wilson, I. B., (1962),J. Biol. Chem., 237, 3245-3249. In this case enzyme and inhibitor arepre-incubated for a set period of time prior to quenching of thisreaction by the addition of an excess of substrate. The second method(Scheme 2), is monitoring enzyme inactivation in the presence ofsubstrate and irreversible inhibitors. This method has been describedpreviously (Tian, W. -X. & Tsou, C. -L, (1982), Biochemistry, 21,1028-1032; Morrison, J. F. & Walsh, C. T., (1988), Adv. Enzymol. Relat.Areas Mol. Biol., 61, 201-301) and the equations describing the kineticsare shown below (Eq. 1, 2 and 3). In both cases the inhibitorconcentration employed is at least 10 times greater than the enzymeconcentration in order to maintain pseudo-first order conditions.

    k.sub.app =k.sub.2 [I]/1+[S]/K.sub.M [I]+K.sub.1)          Eq. 1

    [Product]=v.sub.s t+(v.sub.0 -v.sub.5)[1-exp(-k.sub.app t)]/k.sub.app +dEq. 2

    second order rate constant=(k.sub.app /[I])(1+[S]/K.sub.M) Eq. 3

The apparent inactivation rate constant (k_(app)) was calculated usingEq. 2; where v_(o) is the initial velocity of the reaction, v_(s) isasymptotic steady-state velocity of the reaction, d is the intercept attime zero. The second order rate was calculated using Eq. 3.

Inhibition Kinetics of Der-p I

Assays were routinely carried out in 50 mM potassium phosphate; pH 8.25containing 1 mM ethylenediaminetetraaceticacid (EDTA) and 1 mMdithiothreitol (DTT). The fluorogenic substrate was2-aminobenzoylvalylalanylnorleucylseryl-(3-nitro)tyrosinylaspartylamide. Product formation was monitored with respect to time bymeasuring the increase in fluorescence emission at 420 nm and excitingat 320 nm. Assays were carried out at 25° C. Stock solutions of thevarious inhibitors were made up in 100% dimethylsulphoxide.

Inactivation kinetics for various inhibitors were carried out using thetechniques already described. In the dilution method, generally 100 nMDer p I was mixed and incubated with 0.5-10 uM of the inhibitor andaliquots were taken out at given time points (sampling time) and theresidual enzyme activity determined by a ten-fold dilution into assaybuffer containing saturating amounts of substrate. The residual activitywas related to the sampling time and the curve fitted by computationalnon-linear least square regression analysis. In cases where the secondorder rate constants were greater than 10⁵ M⁻¹ s⁻¹, second orderconditions were employed (i.e. equimolar amounts of enzyme andinhibitor). Generally stoichiometric amounts of enzyme and inhibitorwere incubated for given time intervals (sampling time) and the reactionstopped by a ten-fold dilution of this mixture by saturating amounts ofsubstrate in assay buffer. A plot of reciprocal enzyme concentrationversus sampling time was fitted by linear least square regressionanalysis to obtain the second order inactivation rate constant.

In cases where inactivation kinetics were calculated in the presence ofenzyme, inhibitor and substrate the following conditions were employed.Generally a solution containing 12.5 mM substrate and 0.1-10 mMinhibitor was incubated at 25° C. for 5 min. prior to addition of enzyme(10 nM) to initiate the reaction. In the absence of inhibitor, productformation was linear with time. Inactivation of enzyme was exhibited bythe downward curvature in the increase in fluorescence. The apparentinactivation rate constant (k_(app)) was determined by fitting thesecurves to Eq. 2, using least square regression analysis, and the secondorder rate constant determined using Eq. 3.

Assay Results

    ______________________________________                                               Compound                                                                              k.sub.obs /[I]                                                   number (M.sup.- s.sup.-1)                                                   ______________________________________                                                3      >10.sup.7                                                        4         1.6 × 10.sup.7                                                6         6.8 × 10.sup.7                                                7         3.7 × 10.sup.5                                                8     >10.sup.7                                                               9         2.3 × 10.sup.4                                                10           1.9 × 10.sup.5                                             11           1.2 × 10.sup.6                                             12           1.9 × 10.sup.5                                             13           6.6 × 10.sup.5                                             15           1.5 × 10.sup.5                                             16        1.6 × 10.sup.4                                                23           1.7 × 10.sup.3                                             25           3.1 × 10.sup.3                                             26           4.1 × 10.sup.3                                             27           6.3 × 10.sup.3                                             28           6.8 × 10.sup.3                                             29           4.6 × 10.sup.3                                             30           7.5 × 10.sup.3                                             34           1.1 × 10.sup.4                                             36            6.4 × 10.sup.3                                            38           1.1 × 10.sup.3                                             40           6.9 × 10.sup.4                                           ______________________________________                                    

Compounds for which no inhibition data is shown were key intermediatesin the formation of further compounds or were too unstable to be testedand hence were intermediates to more stable compounds.

Pharmacophore Definition and Specification

A collection of compounds with biological activity for Der p I wasprovided as a training set. Each compound in the training set wassubjected to full conformational analysis (J. Comp. Chem., 1995, 16,171-187). A representative number of conformers were generated over agiven energy range above the lowest energy conformation (J. Chem. Inf.Comp. Sci., 1995, 35, 285-294 and J. Chem. Inf. Comp. Sci., 1995, 35,295-304).

This information was used to derive a pharmacophore (based on sevenchemical feature type rules) (J. Chem. Inf. Comp. Sci., 1994, 34,1297-1308) that correlates to the observed biological activity. It wasassumed that the molecules in the training set all act at the sametarget in the same manner of action.

A pharmacophore consisting of at least the following chemical featuresdefines the chemical motif of potential inhibitors of Der p I:

A Hydrogen bond acceptor feature, three Hydrophobe (J. Comp. Chem.,1986, 7, 565-577) features and a Hydrogen bond donor feature.

A Hydrogen bond acceptor feature matches the following atom types orgroups of atoms which are surface accessible.

sp or sp² nitrogens that have a lone pair of electrons and a charge lessthan or equal to zero

sp³ oxygens or sulphurs that have a lone pair of electrons and chargeless than or equal to zero

non-basic amines that have a lone pair of electrons.

A Hydrogen bond donor feature has the same chemical rules, i.e. itmatches the same atoms or groups of atoms, as the Hydrogen bond acceptorexcept that it also includes basic nitrogen. There is no exclusion ofelectron-deficient pyridines and imidazoles. This feature matches thefollowing atom types or groups of atoms.

non-acidic hydroxyls

thiols

acetylenic hydrogens

NH moieties (except tetrazoles and trifluoromethyl sulfonamidehydrogens).

A Hydrophobe feature is defined as

a contiguous set of atoms that are not adjacent to a concentration ofcharge (charged atoms or electronegative atoms), in a conformation suchthat the atoms have surface accessibility, including phenyl, cycloalkyl,isopropyl and methyl. This may also include residues which have apartial hydrophobic character such as Lysyl or Glutaminyl amino acidside-chains.

The term "pharmacophore" used herein is not meant to imply anypharmacological activity. The term refers to chemical features and theirdistribution in three-dimensional space which constitute and epitomisethe preferred requirements for molecular interaction with a receptor.For example the receptor may be the catalytic active site of thecysteine protease Der p I.

FIG. 4 graphically shows the pharmacophore of Der p I. In the figure theHydrogen bond acceptor is represented by a vector function consisting oftwo spheres. The smaller sphere (at least 1.6 Angstroms radius up to 2.6Angstroms) defines the centroid of the hydrogen bond acceptor on theligand while the large sphere (at least 2.2 Angstroms radius up to 2.6Angstroms) defines the projected point of the hydrogen bond acceptorfrom the receptor. These two spheres are at least 3.0 Angstroms apart.

Similarly the Hydrogen bond donor is represented by a two sphere vectorfunction defined in the same way as above for the Hydrogen bondacceptor.

The Hydrophobe features are represented by spheres of at least 1.6Angstroms radius up to 2.6 Angstroms.

The absolute sphere centroid positions of each feature are defined inthree dimensions as follows:

Hydrophobe 1 has Cartesian XYZ co-ordinates of -6.272, 3.372, -1.200

Hydrophobe 2 has co-ordinates of -3.320, -2.305, 0.906

Hydrophobe 3 has co-ordinates of -0.612, -4.088, -1.740

Hydrogen Bond Donor origin co-ordinates of 0.007, 0.926, 4.168

Hydrogen Bond Donor projected point co-ordinates of -0.743, 0.926, 4.168

Hydrogen bond acceptor origin co-ordinates of 5.155, -0.25, -2.528

Hydrogen bond acceptor projected point co-ordinates of 7.413, 0.349,-4.426

The distance constraints are shown in FIGS. 5 and 10 to 19. The angleconstraints are shown in FIGS. 6 and 10 to 19.

The tolerances on all distances between the chemical features is +/-0.5Angstroms and the geometric angles +/-20 Degrees.

REFERENCES

1. Sutton, B. J. & Gould, H. J. Nature 366, 421-428 (1993).

2. Flores-Romo, L. et al. Science 261, 1038-1041 (1993).

3. Yu, P. et al. Nature 369, 753-756 (1994).

4. Stief, A. et al. J. Immunol. 152, 3378-3390 (1994).

5. Fujiwara, H. et al. Proc. Natl. Acad. Sci. USA 91, 6835-6839 (1994).

6. Chapman, M. D. et al., J. Allergy Clin. Immunol. 72,27-33 (1983).

7. Krillis, S. et al. J. Allergy Clin. Immunol. 74,132-141 (1984).

8. Barrett, A. J. et al. Biochem. J. 201, 189-198 (1982).

9. Mast, A. E. et al. Biochemistry 31, 2720-2728 (1992).

10. Knapp, W. et al. eds. Leucocyte typing IV, Oxford University Press.142-154 (1989).

11. Liu Y. J. et al. Eur. J. Immunol. 21, 1107-1114 (1991).

12. Gordon, J. et al. Immunol. Today 10, 153-157 (1989).

13. Letellier M. et al. J. Exp. Med. 172, 693-700 (1990).

14. Kim, K -M. et al. Pediatric Res. 26, 49-53 (1989).

15. Yanagihara, Y. et al. Clin. Exp. Allergy 20, 395-401 (1990).

16. Chua, K. Y. et al. J. Exp. Med. 167, 175-182 (1988).

17. Finkelman, F. D. & Urban J. F. Parasitol. Today 8, 311-314 (1992).

18. Lombardero, M. et. al. J. Immunol. 144, 1353-1360 (1990).

19. Ghadieri, A. A. et al. Immunol. Lett. 27, 113, (1991).

20. Ghose, A. et al. J. Comp. Chem., 1986, 7, 565-577

21. Smellie, A. et al. J. Comp. Chem., 1995, 16, 171-187

22. Smellie, A. et al. J. Chem. Inf. Comp. Sci., 1995, 35, 285-294

23. Smellie, A. et al. J. Chem. Inf. Comp. Sci., 1995, 35, 295-304

24. Greene, J. et al. J. Chem. Inf. Comp. Sci., 1994, 34, 1297-1308

25. Maeji, N. J. Bray, A. M. Valerio, R. M. and Wang, W., PeptideResearch, 8(1), 33-38, 1995.

26. Valerio, R. M. Bray, A. M. and Maeji, N. J. Int. J. Pept. Prot. Res,44, 158-165, 1994.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 1                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala                                  1               5   - #                10                                   __________________________________________________________________________

We claim:
 1. A compound which has Der p I cysteinyl protease inhibitoractivity and is capable of inhibiting proteolytic cleavage of membranebound CD23 in vivo excludingL-trans-epoxysuccinyl-leucylamido(4-guanidino) butane (E64).
 2. Acomposition capable of adopting a structure essentially equivalent to aninhibitor of the enzyme Der pI, excluding E64, comprising a cysteinylprotease inhibitor compound together with a pharmaceutically acceptablecarrier or excipient for use in the treatment of allergic diseases.
 3. Acysteinyl protease inhibitor compound which adopts a structure having apharmacophoric pattern essentially equivalent to the pharmacophoricpattern of a section of an inhibitor of Der p I, excluding E64.
 4. Aligand which cross reacts with a cysteinyl protease inhibitor compoundwhich inhibits the enzyme Der p I, excluding E64, which compound whichadopts 1 or more copies of a motif which comprises:i) a hydrogen bonddonor; ii) three hydrophobes; and iii) a hydrogen bond acceptor.
 5. Aligand according to claim 4 which comprises a structure essentiallyequivalent to the pharmacophore defined as follows:the pharmacophoreincludes at least 5 chemical features, 3 hydrophobes, a Hydrogen bondacceptor and a Hydrogen Bond Donor; these features being further definedas follows:(1) the hydrogen bond acceptor feature matches the followingatom types or groups of atoms which are surface accessible;sp or sp²nitrogens that have a lone pair and a charge less than or equal to zerosp³ oxygens or sulphurs that have a lone pair and charge less than orequal to zero non-basic amines that have a lone pair; (2) the hydrogenbond donor feature has the same chemical characteristics as the hydrogenbond acceptor except that it also includes basic nitrogen (there is noexclusion of electron-deficient pyridines and imidazoles); this featurematches the following atom types or groups of atoms;non-acidic hydroxylsthiols acetylenic hydrogens NH moieties (except tetrazoles andtrifluoromethyl sulfonamide hydrogens); (3) the hydrophobes are definedas a contiguous set of atoms that are not adjacent to a concentration ofcharge (charged atoms or electronegative atoms), in a conformation suchthat the atoms have surface accessibility, including phenyl, cycloalkyl,isopropyl, methyl and includes residues which have a partial hydrophobiccharacter such as Lysyl or Glutaminyl amino acid side-chains; and(i) thehydrogen bond acceptor is represented by a vector function consisting oftwo spheres; the smaller sphere (at least 1.6 Angstroms radius up to 2.6Angstroms) defines the centroid of the hydrogen bond acceptor on theligand while the large sphere (at least 2.2 Angstroms radius up to 2.6Angstroms) defines the projected point of the hydrogen bond acceptorfrom the receptor; these two spheres are at least 3.0 Angstroms apart;(ii) the hydrogen bond donor is represented by a two sphere vectorfunction as (i) above; (iii) the hydrophobes are represented by spheresof at least 1.6 Angstroms radius up to 2.6 Angstroms; and wherein thetolerances on all distances between these features is +/-0.5 Angstromsand the geometric angles +/-20 Degrees and said distances and angles areshown in FIGS. 4, 5 and 10 to
 19. 6. A composition for treatment of IgEmediated allergic disease which includes as active ingredient aneffective amount of a compound selected from the group consisting of: acysteinyl protease inhibitor; a substrate for Der p I which reacts withDer p I at a specific site; and a Der p I inhibitor capable ofinhibiting the proteolytic enzyme activity of Der p I, the agentoptionally including one or more of a pharmaceutically acceptablecarrier, adjuvant, excipient.
 7. An agent for attenuating orinactivating the allergenicity of Der p I which includes as activeingredient an effective amount of a compound having Der p I inhibitoractivity, the agent optionally including one or more of a carrier,adjuvant, excipient.
 8. A composition for reducing or destroying theviability of house dust mites which includes as active ingredient aneffective amount of a compound having Der p I inhibitor activity, theagent optionally including one or more of a pharmaceutically acceptablecarrier, adjuvant, excipient.
 9. A process for producing a compound orligand according to claim 1 which comprises synthesising a cysteinylprotease inhibitor compound or ligand as defined in any of claims 1 to11 and optionally conjugating said compound or ligand to a carrier. 10.A process according to claim 9 including the further step of isolatingand purifying said compound or ligand.
 11. A pharmaceutical compositioncontaining as active ingredient at least one compound or ligandaccording to claim 1 and optionally including an adjuvant or excipient.12. A pharmaceutical composition according to claim 11 for use in thetreatment of an IgE-mediated allergic disease.
 13. A pharmaceuticalcomposition according to claim 11 for use in prophylactic prevention ofjuvenile asthma or eczema.
 14. A pharmaceutical composition according toclaim 11 for use in the treatment of juvenile asthma or eczema.